It is known that droplets with a volume of more than 10 μl can be dispensed from the air very easily, since if the pipette is correctly manipulated, the droplets leave the pipette tip of their own accord. The droplet size is then determined by the physical properties of the sample liquid, such as surface tension or viscosity. The droplet size thus limits the resolution of the quantity of liquid to be dispensed.
The aspirating and dispensing, i.e. the pipetting of liquid samples with a volume of less than 10 μl, in contrast, typically requires instruments and techniques which guarantee the dispensing of such small samples. The dispensing of a liquid with a pipette tip, i.e. with the endpiece of a device for aspirating and/or dispensing sample liquid, can occur from the air (“from air”) or by touching a surface. This surface can be the solid surface of a container (“on tip touch”), into which the liquid sample is to be dispensed. It can also be the surface of a liquid in this container (“on liquid surface”). A mixing procedure following the dispensing is recommended—particularly for very small sample volumes in the nanoliter or even picoliter range—so that uniform distribution of the sample volume in a diluent is ensured.
Disposable tips significantly reduce the danger of unintentional transfer of parts of the sample (contamination). Simple disposable tips are known (so-called “air-displacement tips”), whose geometry and material is optimized for the exact aspirating and dispensing of very small volumes. The use of so-called “positive-displacement tips”, which have a pump plunger inside, is also known.
For automation of the pipetting process, two procedures must be differentiated from one another: the defined aspiration and the subsequent dispensing of liquid samples. Between these procedures, typically the pipette tip is moved by the experimenter or by a robot, so that the aspiration location of a liquid sample is different from its dispensing location. For the precision of aspiration and dispensing, only the liquid system is essential, which includes a pump (e.g. a diluter implemented as a syringe pump), tubing, and an endpiece (pipette tip). Among the many possible pumps for highly precise aspirating and dispensing of liquids, commercially available devices with the name “CAVRO XL 3000 Modular Digital Pump” or “CAVRO XP3000 plus Modular Digital Pump”, sold by the firm Tecan Systems Inc., 2450 Zanker Road, San Jose, Calif. 95131 USA (formerly Cavro Scientific Instruments Inc., Sunnyvale, Calif., USA), have, for example, proven themselves. Such pumps include a cylinder with a piston movable therein and a stepping motor for driving the piston. The stepping motor operates at a voltage of 24 V and is controlled by an external computer or microprocessor. Further details can, for example, be found in the “Operators Manual P/N 724043C” from Cavro Scientific Instruments Inc.
A device and a corresponding method are known from U.S. Pat. No. 5,763,278. They involve automatic pipetting of small volumes, with the device including a pipetting needle, a diluter with a liquid outlet having a syringe, and a valve. The syringe includes a piston and a piston drive. A line connects the needle and the liquid outlet of the diluter, with the diluter and the line containing an essentially incompressible liquid. A pulse generator is located in the device and connected with the incompressible liquid in the line so that mechanical pulses with a force of at least 0.01 Ns can be output directly into the liquid of the line. A pulse of this type serves for driving the liquid out of the needle. The droplet size is defined by a targeted advance of the diluter piston and the droplet is ejected from the needle with a pulse. By defining the volume with the diluter, the droplet size and its reproducibility depends on the resolution of the diluter and is limited by it. Another pipetting device of this class which includes a piston pump and a pulse generator in the form of a piezoelectric element is known from JP 09 327628.
Multichannel systems in which 4, 8, or 12 pipetting channels are distributed on one line have been known for some time. The tips are either arranged in a fixed raster (e.g. the MiniPrep device series from the firm CAVRO Scientific Instruments Inc., Sunnyvale, Calif., USA) or they can be spread out along one line (e.g. the GENESIS device series from TECAN Schweiz AG, Seestrasse 103, CH-8708 Männedorf). The pipetting channels are either operated jointly via a stepping motor with one or more syringes or individually operated via the same number of diluters as syringes.
Multichannel systems for volumes in the sub-microliter range are known in the form of fixed two-dimensional combs (e.g. the PixSys4500 from Cartesian Technologies, Inc., 17851 Sky Park Circle, Irvine, Calif. 92614, USA) or from EP 0 956 449. These two-dimensional combs of pipettes, however, are typically no longer sufficient for the current demands for sample throughput.
Multichannel pipettors arranged in three dimensions are also known. They can be implemented as 96 tip devices with 96 individual hoses and 96 individual syringes, which are each driven in groups of 8 by a joint stepping motor (e.g. the MICROLAB MPH-96 Workstation from Hamilton Bonaduz AG, P.O. Box 26, 7402 Bonaduz, Switzerland). This system is very costly due to the large number of syringes and motors. In addition, it is difficult to remove interfering air bubbles from all of the hoses.
Arrays with up to 384 individual glass syringes with cannulas have also been arranged in the raster of a 384 microplate. The plungers of the syringes are moved simultaneously by one single stepping motor (e.g. the Hydra from Robbins Scientific, 1250 Elko Drive, Sunnyvale, Calif. 94089-2213, USA). The method is costly due to the many syringes. It cannot be expanded for disposable tips.
In place of diluters, syringes, and pistons, metal bellows are also used (cf. U.S. Pat. No. 5,638,986). Due to the smaller mass to be moved, dispensing speeds are achieved which are suitable for dispensing volumes down to 0.5 μl from the air (e.g. the Liliput Dispenser from Fluilogic Systems Oy, Luoteisrinne 4, 02270 Espoo, Finland). A disadvantage is, however, that the metal bellows cannot be calibrated like, for example, a diluter.
The most frequent constructional principle of three-dimensionally arranged multichannel pipettors comprises a plate to which and/or in which the 96 or 384 pistons or plungers are attached. As known from U.S. Pat. No. 4,087,248, this plate is moved, with the pistons for aspirating and/or dispensing, up and down by one or more motors. In U.S. Pat. No. 4,087,248 (see there FIG. 3), feeding reagents, solutions, and the like, to the tips of syringes is described: According to one embodiment, the syringes are in fluid connection with a reservoir through orifices. When the plunger is moved above the orifices, fluid from the reservoir will enter the syringe. This fluid may then be expelled from the syringe into the tips by moving the plunger downwardly. In an alternative embodiment, a so-called “Eppendorf Syringe”, comprising a hollow plunger with a central post, is employed. The central post is spring mounted and connected to a foot. An O-ring is compressed between the foot and a projection which is rigidly attached to the plunger. The foot is shaped to leave a gap between the foot and the inner wall of the syringe. The O-ring provides a seal with the inner wall of the syringe. By pressing on the central post, the foot is lowered with respect to the projection and O-ring so as to provide fluid connection between the hollow center of the plunger and the syringe. Fluid can therefore dispensed by means of the plunger or withdrawn from the syringe employing suction. On the one hand, the alternative embodiment with the “Eppendorf Syringes” appears to be too complex in particular for a multichannel pipetter with 96 or more pipetting channels. On the other hand, the first embodiment suffers from the fact that the plunger has to be moved against the pressure of the rinsing or flushing liquid for aspiration of a liquid sample; the same pressure acting with the plunger for dispensing liquid samples. Such pressure differences provoke a complex drive and monitoring system, if accurate pipetting is targeted.